Human development depends on the development and function of the placenta. This organ connects the mother to her fetus providing transport of oxygen and nutrients and serves as a master regulator of pregnancy. Understanding human placenta development and function has been challenging in that placental structure and development is not highly conserved across mammalian species and longitudinal analysis of on-going human pregnancies is limited by practical and ethical considerations. The placenta releases a variety of small membrane bound particles into the maternal circulation. These placental packages provide an opportunity to assess the functional state of the placenta. This project seeks to develop a novel tool to monitor one type of these package types, exosomes, as the cargo reflects the physiologic state of the tissue of origin. The approach is to extend the capabilities of CyTOF (a tandem flow cytometry and mass spectroscopy technology) from the single-cell level of interrogation to the single-vesicle level to analyze placental-specific exosomes from maternal circulation to reveal the functional state of the human placenta. This will be done through the following aims. (1) Develop and validate a novel tool (P-Exo-CyTOF) to identify human placental-specific exosomes using the CyTOF platform. Innovative metal-based vesicle labeling, specific metal-tagged antibody panels for placental exosomes and novel computational workflows will be developed and optimized to enable the detection of placenta-derived exosomes by CyTOF. Exosomes derived from human trophoblast cell lines cultured in various oxygen tensions as well as maternal plasma from normotensive and pre-eclamptic pregnancies will be used to validate and test the sensitivity, specificity and utility of P-Exo-CyTOF in proof of principle experiments. (2) Determine exosome markers that reflect the gestational and functional state of the human placenta. Using semi-quantitative mass spectrometry, the proteome of human placental exosomes over gestation and in the context of preeclampsia will be determined. These studies will inform the composition of comprehensive functional P-Exo-CyTOF antibody panels representative of placental cellular states. Not only will this proposal advance the capabilities of CyTOF in terms of extracellular vesicle assessment but will provide the ability to safely study and monitor the human placenta in real-time during an on-going pregnancy.